This invention relates to compounds and methods for inducing or promoting apoptosis and for arresting uncontrolled neoplastic cell proliferation, methods that are specifically useful in the arresting and treatment of neoplasias, including precancerous and cancerous lesions.
Pharmaceuticals that are effective against early stage neoplasias comprise an emerging and expanding area of research and potential commercial development. Such pharmaceuticals can delay or arrest development of precancerous lesions into cancers. Each year in the United States alone, untold numbers of people develop precancerous lesions, which exhibit a strong statistically significant tendency to develop into malignant tumors, or cancer. Such lesions include lesions of the breast (that can develop into breast cancer), lesions of the skin (that can develop into malignant melanoma or basal cell carcinoma), colonic adenomatous polyps (that can develop into colon cancer), cervical dysplasia (cervical cancer) and other such neoplasms.
Such compounds and methods are particularly beneficial to sub-populations of patients who repeatedly develop precancerous lesions, and therefore have a statistically higher probability of getting cancer. Many cancer types (e.g., breast, colon, prostate etc.) have such patient sub-populations.
The search for drugs useful for treating and preventing neoplasias in their earliest stages is intensive because chemotherapy and surgery on cancer itself is often not effective, and current cancer chemotherapy has severe side effects. Such cancer-preventative compounds are also envisaged for recovered cancer patients who retain a risk of cancer reoccurrence, and even for cancer patients who would benefit from compounds that selectively induce apoptosis in neoplastic, but substantially not in normal cells.
Because it is believed that chronic administration of cancer-preventative pharmaceuticals is necessary to inhibit or arrest the development of neoplasia, standard cancer chemotherapeutic drugs are not considered appropriate drugs for cancer chemoprevention because, whatever cancer preventative (as opposed to cancer-fighting) capabilities those drugs may possess, they do not outweigh their severe side effects. Most standard chemotherapeutics are now believed to kill cancer cells by inducing apoptosis (also sometimes referred to as xe2x80x9cprogrammed cell deathxe2x80x9d). Apoptosis naturally occurs in many tissues in the body. Apoptosis plays a critical role in tissue homeostasis, that is, it ensures that the number of new cells produced are correspondingly offset by an equal number of cells that die. Apoptosis is especially pronounced in self-renewing tissues such as bone marrow, immune cells, gut, and skin. For example, the cells in the intestinal lining divide so rapidly that the body must eliminate cells after only three days to protect and prevent the overgrowth of the intestinal lining.
Standard chemotherapeutics promote apoptosis not only in cancer cells, but also in normal human tissues, and therefore have a particularly severe effect on tissues where apoptosis is especially pronounced (e.g. hair, gut and skin). The results of those effects include hair loss, weight loss, vomiting and bone marrow immune suppression. Thus, standard chemotherapeutics are inappropriate for cancer prevention, particularly if chronic administration is indicated.
Several non-steroidal anti-inflammatory drugs (xe2x80x9cNSAIDsxe2x80x9d), originally developed to treat arthritis, have shown effectiveness in inhibiting and eliminating colonic polyps. Polyps virtually disappear when the patients take the drug, particularly when the NSAID sulindac is administered. However, the continued prophylactic use of currently available NSAIDs, even in high colon cancer-risk patients, is still marked by severe side reactions that include gastrointestinal irritations, perforations, ulcerations and kidney toxicity believed to be produced by inhibition of prostaglandin synthetase activity (xe2x80x9cPGE-2xe2x80x9d). Such inhibition is a requirement for the NSAIDs anti-inflammatory action since elevated levels of PGE-2 are associated with inflammation. PGE-2 plays a protective function in the gastrointestinal tract, which is the reason such gastric side effects arise with chronic NSAID therapy, which is rarely indicated for arthritis sufferers, acute therapy being the norm for them. However, chronic administration of sulindac is important for high cancer-risk patients to eliminate and prevent future polyps which cause gastric side effects in many such patients. Once NSAID treatment is terminated due to such complications, the neoplasms return, particularly in high risk patients.
Compounds such as those disclosed in U.S. Pat. No. 5,643,959 have exhibited advantages in the treatment of neoplastic lesions since such compounds have been shown to induce apoptosis in neoplastic cells but not in normal cells in humans (see Piazza et al. Gastroenterology Vol. 112, A629, 1997). Thus, the severe side effects due to induction of apoptosis in normal cells by conventional chemotherapeutics are avoided by these novel therapeutics (see, Piazza et al. Cancer Research Vol. 57, pp. 2452-2459, 1997). In addition, such compounds do not exhibit the gastric side effects associated with NSAIDs since such compounds do not substantially inhibit PGE-2. More potent compounds with such neoplasia specificity but without substantial PGE-2 activity are desirable.
This invention represents potent compounds that inhibit the growth of neoplastic cells, for treating patients with neoplastic lesions without substantially inhibiting PGE-2. This invention also involves methods for inducing such specific inhibition of neoplastic cells by exposing such cells to a pharmacologically effective amount of those compounds described below to a patient in need of such treatment. Such compositions are effective in modulating the growth of neoplasms.
As discussed above, the present invention includes compounds of Formula I below (as well as their pharmaceutically acceptable salts) for treating a patient with neoplastic, particularly precancerous, and cancerous lesions: 
wherein
Y1 and Y2 are independently selected from the group consisting of (CH2)n, xe2x80x94C(X)xe2x80x94NHxe2x80x94, xe2x80x94(CH2)nxe2x80x94C(X)xe2x80x94Oxe2x80x94, xe2x80x94C(X)xe2x80x94Oxe2x80x94 and X is O or S;
R1 is selected from the group consisting of lower alkyl, lower alkenyl, lower alkynyl, hydrogen, and substituted or unsubstituted phenyl, pyridinyl, pyrrolyl, pyrrolidinyl, pyrazolyl, pyrazolidinyl, imidazolyl, imidazolidinyl, piperidinyl, pyrazinyl, piperazinyl, pyzimidinyl, naphthyl, morpholinyl, tetrazolyl, triazinyl, furfulyl and thiophenyl, wherein said substituents are one to three independently selected from the group consisting of halogen, lower alkyl, lower alkoxy, amino, lower alkylamino, di-lower alkylamio, hydroxy, nitro, nitrile, carboxyl, sulfonylamido, lower alkyl mercapto, and lower alkyl sulfonyl;
R2 is selected from the group consisting of substituted or unsubstituted phenyl, benzyl, pyridinyl, pyrrolyl, pyrrolidinyl, pyrazolyl, pyrazolidinyl, imidazolyl, imidazolidinyl, piperidinyl, pyrazinyl, piperazinyl, pyrimidinyl, tetrazolyl, morpholinyl, triazinyl, furfuryl and thiophenyl, wherein said substituents are one to three independently selected from the group consisting of halogen, lower alkyl, lower alkoxy, amino, lower alkylanmino, di-lower alkylamino, hydroxy, nitro, nitrile, carboxyl, aminosulfonyl, lower alkyl mercapto, and lower alkyl;
A is a ring fused with the pyridazine ring selected from the group consisting of benzene, pyndine, pyrrol, pyrtolidine, pyrazol, pyrazolidine, imidazol, imidazolidine, piperidine, pyrazine, piperazine, pyrimidine, morpholine, tetrazol, triazine, furan and thiophene; piperidine, pyrazine, piperazine, pyrimidine, morpholine, tetrazol, triazine, furan and thiophene;
(R3) are substituents replacing hydrogen on the ring fused to pyridazine. R3 is independently selected in each instance from the group consisting of halogen, lower alkyl, lower alkoxy, amino, lower alkylamino, di-lower alkylamino, hydroxy, nitro, nitrile, carboxyl, sulfonylamido, lower alkyl mercapto, and lower alkyl sulfonyl;
m is an integer from 1 to 4;
n is an integer from 0 to 3
p is 0 or 1;
q is 0, 1 or 2; and
pharmaceutically acceptable salts thereof.
Preferred compounds of this invention include those where (1) R1 is selected from the group consisting of lower alkyl, lower alkenyl, lower alkynyl, and substituted or unsubstituted phenyl, pyridinyl, pyrrolyl, pyrazolyl, imidazolyl, pyrazinyl, pyrimidinyl, morpholinyl, triazinyl, furfuryl and thiophenyl, wherein said substituents are one or two independently selected from the group consisting of halogen, lower alkoxy, di-lower alkylamino, hydroxy, nitrile, carboxyl, sulfonylamido, lower alkyl mercapto, and lower alkyl sulfonyl; (2) R2 is selected from the group consisting of substituted or unsubstituted phenyl, benzyl, pyridinyl, pyrrolyl, pyrazolyl, imidazolyl, pyrazinyl, pyrimidinyl, morpholinyl, triazinyl, furfuryl and thiophenyl, wherein said substituents are one or two independently selected from the group consisting of halogen, lower alkyl, lower alkoxy, di-lower alkylamino, hydroxy, carboxyl, aminosulfonyl, lower alkyl mercapto, and lower alkyl; (3) xe2x80x9cAxe2x80x9d is a ring fused with the pyridazine ring selected from the group consisting of benzene, pyridine, pyrrol, pyrrolidine, imidazolidine, piperidine, pyrazine, piperazine, pyrimidine, morpholine, triazine, furan and thiophene; (4) p is 0 or 1; (5) R3 is independently selected in each instance from the group consisting of halogen, lower alkyl, lower alkoxy, di-lower alkylamino, hydroxy, carboxyl, sulfonylamido, lower alkyl mercapto, and lower alkyl sulfonyl; and m is an integer from 0 to 2; and (6) n is an integer from 0 to 2.
More preferred compounds of this invention include those wherein (1) R1 is selected from the group consisting of substituted or unsubstituted phenyl, pyridinyl, pyrazinyl, pyrimidinyl, triazinyl, furfuryl and thiophenyl, wherein said substituent is one member selected from the group consisting of halogen, lower alkoxy, di-lower alkylamino, nitrile, sulfonylamido, and lower alkyl sulfonyl; (2) R2 is selected from the group consisting of substituted or unsubstituted phenyl, benzyl, pyridinyl, pyrazinyl, pyrimidinyl, triazinyl, furfuryl and thiophenyl, wherein said substituent is one member selected from the group consisting of halogen, lower alkyl, lower alkoxy, di-lower alkylamino, aminosulfonyl, and lower alkyl mercapto; (3) wherein xe2x80x9cAxe2x80x9d is a ring fused with the pyridazine ring selected from the group consisting of benzene, pyrazine, pyrimidine, furan and thiophene; (4) p is 0; (5) R3 is independently selected in each instance from the group consisting of halogen, lower alkyl, lower alkoxy, di-lower alkylamino, and lower alkyl mercapto; and m is 0 or 1; and (6) n is 0 or 1.
The present invention is also a method of treating individuals with neoplastic lesions by administering a pharmacologically effective amount of an enterically coated pharmaceutical composition that includes compounds of this invention.
Preferably, such compounds are administered without therapeutic amounts of an NSAID.
Also, the present invention is a method of inhibiting the growth of neoplastic cells by exposing the cells to an effective amount of compounds of Formula I
As used herein, the term xe2x80x9cprecancerous lesionxe2x80x9d includes syndromes represented by abnormal neoplastic, including dysplastic, changes of tissue. Examples include dysplasic growths in colonic, breast, bladder or lung tissues, or conditions such as dysplastic nevus syndrome, a precursor to malignant melanoma of the skin. Examples also include, in addition to dysplastic nevus syndromes, polyposis syndromes, colonic polyps, precancerous lesions of the cervix (i.e., cervical dysplasia), esophagus, prostatic dysplasia, bronchial dysplasia, breast, bladder and/or skin and related conditions (e.g., actinic keratosis), whether the lesions are clinically identifiable or not.
As used herein, the term xe2x80x9ccancerousxe2x80x9d refers to lesions that are malignant. Examples include malignant melanomas, breast cancer, prostate cancer and colon cancer.
As used herein, the term xe2x80x9cneoplasmxe2x80x9d refers to both precancerous and cancerous lesions and hyperplasia.
As used herein, the term xe2x80x9chaloxe2x80x9d or xe2x80x9chalogenxe2x80x9d refers to chloro, bromo, fluoro and iodo groups, and the term xe2x80x9calkylxe2x80x9d refers to straight, branched or cyclic alkyl groups and to substituted aryl alkyl groups. The term xe2x80x9clower alkylxe2x80x9d refers to C1 to C8 alkyl groups.
The term xe2x80x9clower alkoxyxe2x80x9d refers to alkoxy groups having from 1 to 8 carbons, including straight, branched or cyclic arrangements.
The term xe2x80x9clower alkylmercaptoxe2x80x9d refers to a sulfide group that is substituted with a lower alkyl group; and the term xe2x80x9clower alkyl sulfonylxe2x80x9d refers to a sulfone group that is substituted with a lower alkyl group.
The term xe2x80x9cpharmaceutically acceptable saltxe2x80x9d refers to non-toxic acid addition salts and alkaline earth metal salts of the compounds of Formula I. The salts can be prepared in situ during the final isolation and purification of such compounds, or separately by reacting the free base or acid functions with a suitable organic acid or base, for example. Representative acid addition salts include the hydrochloride, hydrobromide, sulfate, bisulfate, acetate, valerate, oleate, palmetate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, mesylate, citrate, maleate, fumarate, succinate, tartrate, glucoheptonate, lactobionate, lauryl sulfate salts and the like. Representative alkali and alkaline earth metal salts include the sodium, calcium, potassium and magnesium salts.
It will be appreciated that certain compounds of Formula I can possess an asymmetric carbon atom and are thus capable of existing as enantiomers. Unless otherwise specified, this invention includes such enantiomers, including any racemates. The separate enantiomers may be synthesized from chiral starting materials, or the racemates can be resolved by conventional procedures that are well known in the art of chemistry such as chiral chromatography, fractional cyrstallization of diastereomeric salts and the like.
Compounds of this invention may be formulated into pharmaceutical compositions together with pharmaceutically acceptable carriers for oral administration in solid or liquid form, or for intraveneous, rectal or topical administration, although carriers for oral administration are most preferred.
Pharmaceutically acceptable carriers for oral administration include capsules, tablets, pills, powders, troches and granules. In such solid dosage forms, the carrier can comprise at least one inert diluent such as sucrose, lactose or starch. Such carriers can also comprise, as is normal practice, additional substances other than diluents, e.g., lubricating agents such as magnesium stearate. In the case of capsules, tablets, troches and pills, the carriers may also comprise buffering agents. Carriers such as tablets, pills and granules can be prepared with enteric coatings on the surfaces of the tablets, pills or granules. Alternatively, the enterically coated compound can be pressed into a tablet, pill, or granule for administration to the patient. Preferred enteric coatings include those that dissolve or disintegrate at colonic pH such as shellac or Eudraget S.
Pharmaceutically acceptable carriers include liquid dosage forms for oral administration, e.g., pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs containing inert diluents commonly used in the art, such as water. Besides such inert diluents, compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring and perfuming agents.
Pharmaceutically acceptable carriers for topical administration include DMSO, alcohol or propylene glycol and the like that can be employed with patches or other liquid-retaining material to hold the medicament in place on the skin so that the medicament will not dry out.
Pharmaceutically acceptable carriers for rectal administration are preferably suppositories that may contain, in addition to the compounds of this invention excipients such as cocoa butter or a suppository wax, or gel.
The pharmaceutically acceptable carrier and compounds of this invention are formulated into unit dosage forms for administration to a patient. The dosage levels of active ingredient (i.e., compounds of this invention) in the unit dosage may be varied so as to obtain an amount of active ingredient effective to achieve lesion-eliminating activity in accordance with the desired method of administration (i.e., oral or rectal). The selected dosage level therefore depends upon the nature of the active compound administered, the route of administration, the desired duration of treatment, and other factors. If desired, the unit dosage may be such that the daily requirement of the active compound is in one dose, or divided among multiple doses for administration, e.g., two to four times per day.
The pharmaceutical compositions of this invention are preferably packaged in a container (e.g., a box or bottle, or both) with suitable printed material (e.g., a package insert) containing indications, directions for use, etc.
The general scheme for producing compounds useful in this invention is illustrated and explained on the following page. 
A xcex3-ketocarboxylic acid (b) can be obtained from a substituted or unsubstituted anhydride (a) (reaction 1) by a Friedel-Crafts reaction (OS, CV-I, 517, 1941) with a substituted or unsubstituted aromatic compound (R2xe2x80x94H, method A) or by reaction with a Grignard reagent Hal-(Mgxe2x80x94(Y2)pxe2x80x94R2), followed by acidic workup (method B).
Condensation of acid (b) with hydrazine leads to the annellated pyridazine derivative (c) (reaction 2). The hydroxy phthalazine (c) is converted to the thiophthalazine derivative (d) by stirring with Lawesson""s reagent for 2 hours at (80-90xc2x0 C.) (reaction 3). After treatment with base (e.g., KOH), a SN-reaction with a substituted alkyl or arylhalide generates the product (e)(reaction 4). Oxidation of (e) with oxone(copyright) gives the sulfone derivative (f) (reaction 5) where q=2.). Oxidation of (e) with H2O2 gives the sulfoxide derivative (g) (reaction 5) where q=1.
A number of xcex3-ketocarboxylic acids (b) are commercially available (e.g., from Aldrich Chemical Co.)
2-benzoylbenzoic acid
2[4-(dibutylamino)-2-hydroxybenzoyl]benzoic acid
3-benzoyl-2-pyridine carboxylic acid
2-(4-fluorobenzoyl)benzoic acid
2-aminobenzophenone-2xe2x80x2-carboxylic acid 
2(3-amino-4-chlorobenzoyl)benzoic acid
4-[4-(2-carboxybenzoyl)phenyl]butyric acid
2-(4-chlorobenzoyl)benzoic acid
In summary, the reagents and conditions for the general schemes are as follows:
(1)
Method A: Friedel-Crafts Reaction R2xe2x80x94H and AlCl3 as a catalyst (p=0).
Method B: Grignard Reaction Hal-Mgxe2x80x94(Y2)pxe2x80x94R2, acid (p=0 or 1).
(2) NH2NH2 
(3) Lawesson""s reagent (Aldrich), toluene, 80-90xc2x0 C.
(4)
a) KOH
b) Hal-Y1xe2x80x94R1 
(5) Oxone(copyright) (Aldrich) DMA or H2O2